HUMANIZED MURINE MODEL OF CHRONIC HEPATITIS B CONSTRUCTED USING STEM CELLS AND METHOD OF USING SAME

Abstract

A humanized murine model of chronic hepatitis B constructed using human stem cells, obtained by obtaining the human stem cells; transplanting the human stem cells into a murine with liver damage to achieve presence of 110.sup.4-8 of the human stem cells in the murine in vivo; etc. The humanized murine model may be used for the study of drug resistance to the antiviral drugs, the study of the pathogenesis of chronic hepatitis B, etc. In addition to the study of the mechanism of hepatitis B viral infection, the humanized murine model may also be used for research in treatment.

Claims

1. A humanized murine model of chronic hepatitis B constructed using human stem cells, wherein the humanized murine model is obtained by the following steps: (a) obtaining the human stem cells; (b) transplanting the human stem cells into a murine with liver damage to achieve presence of 110.sup.4-8 of the human stem cells in the murine in vivo; (c) forming chimera of human-derived hepatocytes, wherein the human stem cells transplanted in the step (b) differentiate into the human-derived hepatocytes in a liver of the murine; and forming a humanized immune system, wherein the human stem cells transplanted in the step (b) differentiate and form the human-derived immune system in the murine in vivo; (d) infecting the murine with hepatitis B virus (HBV); and (e) confirming a HBV infection and a liver disease in the murine.

2. The humanized murine model according to claim 1, wherein the human stem cells originated from stem cells isolated and cultured from a healthy human, or a commercialized cell line.

3. The humanized murine model according to claim 1, wherein the liver damage comprises chemical liver damage caused by a chemical drug, physical liver damage caused by surgery or both; wherein the murine is a normal mouse, an immunodeficient mouse, a normal rat, or an immunodeficient rat.

4. The humanized murine model according to claim 1, wherein in the step (c), the human stem cells which differentiate into the human-derived hepatocytes express a human hapatocytic marker selected from the group consisting of HSA, HLA, ALB and NTCP.

5. The humanized murine model according to claim 1, wherein in the step (c), the human-derived immune system formed by the human by the human stem cells is capable of detecting an immune cell expressing a human lymphocytic marker selected from the group consisting of CD45.sup.+, CD4.sup.+, CD8.sup.+, CD3.sup.+, CD19.sup.+, CD20.sup.+, CD68.sup.+and NKp46.sup.+.

6. The humanized murine model according to claim 1, wherein in the step (e), the HBV infection is confirmed by detecting intact HBV particles in persistent and stable presence in the murine in vivo; wherein the liver disease is selected from the group consisting of hepatitis, liver fibrosis developed from the hepatitis, liver cirrhosis developed from the hepatitis, and liver tumors developed from the hepatitis.

7. The humanized murine model according to claim 2, wherein in the step (e), the HBV infection is confirmed by detecting intact HBV particles in persistent and stable presence in the murine in vivo; wherein the liver disease is selected from the group consisting of hepatitis, liver fibrosis developed from the hepatitis, liver cirrhosis developed from the hepatitis, and liver tumors developed from the hepatitis.

8. The humanized murine model according to claim 3, wherein in the step (e), the HBV infection is confirmed by detecting intact HBV particles in persistent and stable presence in the murine in vivo; wherein the liver disease is selected from the group consisting of hepatitis, liver fibrosis developed from the hepatitis, liver cirrhosis developed from the hepatitis, and liver tumors developed from the hepatitis.

9. The humanized murine model according to claim 4, wherein in the step (e), the HBV infection is confirmed by detecting intact HBV particles in persistent and stable presence in the murine in vivo; wherein the liver disease is selected from the group consisting of hepatitis, liver fibrosis developed from the hepatitis, liver cirrhosis developed from the hepatitis, and liver tumors developed from the hepatitis.

10. The humanized murine model according to claim 5, wherein in the step (e), the HBV infection is confirmed by detecting intact HBV particles in persistent and stable presence in the murine in vivo; wherein the liver disease is selected from the group consisting of hepatitis, liver fibrosis developed from the hepatitis, liver cirrhosis developed from the hepatitis, and liver tumors developed from the hepatitis.

11. The humanized murine model according to claim 6, wherein the hepatitis is confirmed when a symptom comprising punctate necrosis is present in the liver of the murine; the liver fibrosis is confirmed when inflammation and fibrosis are present in the liver of the murine; the liver cirrhosis is confirmed when a characteristic of the liver cirrhosis comprising change in pseudo lobule is found; the liver tumors is confirmed when a lesion of a primary liver tumors observable by histology and imaging is present in the murine in vivo.

12. The humanized murine model according to claim 7, wherein the hepatitis is confirmed when a symptom comprising punctate necrosis is present in the liver of the murine; the liver fibrosis is confirmed when inflammation and fibrosis are present in the liver of the murine; the liver cirrhosis is confirmed when a characteristic of the liver cirrhosis comprising change in pseudo lobule is found; the liver tumors is confirmed when a lesion of a primary liver tumors observable by histology and imaging is present in the murine in vivo.

13. The humanized murine model according to claim 8, wherein the hepatitis is confirmed when a symptom comprising punctate necrosis is present in the liver of the murine; the liver fibrosis is confirmed when inflammation and fibrosis are present in the liver of the murine; the liver cirrhosis is confirmed when a characteristic of the liver cirrhosis comprising change in pseudo lobule is found; the liver tumors is confirmed when a lesion of a primary liver tumors observable by histology and imaging is present in the murine in vivo.

14. The humanized murine model according to claim 9, wherein the hepatitis is confirmed when a symptom comprising punctate necrosis is present in the liver of the murine; the liver fibrosis is confirmed when inflammation and fibrosis are present in the liver of the murine; the liver cirrhosis is confirmed when a characteristic of the liver cirrhosis comprising change in pseudo lobule is found, normal central-portal relationships is lossed; the hepatocellular tumors is confirmed when a lesion of primary liver tumors observable by tissue biopsy and imaging is present in the murine in vivo.

15. The humanized murine model according to claim 10, wherein the hepatitis is confirmed when a symptom comprising punctate necrosis is present in the liver of the murine; the liver fibrosis is confirmed when inflammation and fibrosis are present in the liver of the murine; the liver cirrhosis is confirmed when a characteristic of the liver cirrhosis comprising change in pseudo lobule is found; the liver tumors is confirmed when a lesion of primary liver tumors observable by histology and imaging is present in the murine in vivo.

16. A method of researching and developing an antiviral drug, an anti-fibrosis drug, or an anti-tumor drug, comprising: screening and testing the antiviral drug, anti-fibrosis drug, or anti-tumor drug using the humanized murine model according to claim 1.

17. A method of studying drug resistance to an antiviral drug, comprising: testing the drug resistance to the antiviral drug using the humanized murine model according to claim 1.

18. A method of studying pathogenesis of chronic hepatitis B, comprising: analyzing the pathogenesis of the chronic hepatitis B using the humanized murine model according to claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG. 1 is a graph for the research and development of new antiviral drugs using a humanized mouse model of chronic hepatitis B.

[0033] FIG. 2 is a graph for the study of drug resistance to the antiviral drugs using a humanized rat model of chronic hepatitis B.

[0034] FIG. 3 is a schematic diagram of the study of the pathogenesis of chronic hepatitis B using a humanized rat model of chronic hepatitis B.

[0035] FIG. 4 is a graph for the research and development of new anti-fibrosis drugs using a humanized rat model of liver fibrosis and liver cirrhosis.

[0036] FIG. 5 is a schematic diagram of the study of the pathogenesis of chronic hepatitis B primary liver tumors using a humanized mouse model of hepatitis B primary liver tumors.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0037] The present invention discloses a humanized murine model of chronic hepatitis B constructed based on stem cell technology. The technical solution of the present invention is further explained below:

[0038] A humanized murine model of chronic hepatitis B constructed using stem cells, wherein the murine model is obtained through the following steps: [0039] 1) obtaining human stem cells, wherein the origin of the human stem cells are stem cells isolated and cultured from healthy human, or commercialized cell line; [0040] 2) transplanting the human stem cells into a murine with liver damage such that 110.sup.4-8 of the transplanted human stem cells are present in the murine in vivo; wherein the murine liver damage includes chemical liver damage caused by chemical drugs and physical liver damage caused by surgery; wherein the murine is a normal mouse, an immunodeficient mouse, a normal rat, or an immunodeficient rat; [0041] 3) chimera of human-derived hepatocytes: the transplanted human stem cells differentiate into human-derived hepatocytes in the murine liver, wherein the stem cells may express human hapatocytic markers being HSA, HLA, ALB, NTCP, or the like; [0042] 4) humanized immune system: the transplanted human stem cells differentiate and form a human-derived immune system in the murine in vivo (liver, blood, spleen, bone marrow, etc.), wherein the human-derived immune system differentiated and formed from the transplanted stem cells may detect immune cells expressing human lymphocytic markers including CD45.sup.+/CD4.sup.+/CD8.sup.+/CD3.sup.+/CD19.sup.+/CD20.sup.+/CD68.sup.+/NKp46.sup.+ and the like; [0043] 5) infecting the humanized murine with HBV; [0044] 6) confirming the HBV infection by analyzing HBV DNA, HBsAg, HBeAg, HBcrAg

[0045] HBsAb, HBeAb and HBcAb in serology, HBV cccDNA in hepatocytes and detecting intact HBV particles in persistent and stable presence in the murine in vivo; and [0046] 7) confirming the liver disease: hepatitis is confirmed when symptoms such as punctate necrosis are present in the liver of the murine model; liver fibrosis is confirmed when inflammation and fibrosis are present in the liver of the murine model; liver cirrhosis is confirmed when characteristics of liver cirrhosis such as change in pseudo lobule are found; liver tumors is confirmed when lesions of primary liver tumors observable by histology and imaging are present in the murine model in vivo.

[0047] The humanized murine model of chronic hepatitis B constructed using stem cells according to the present invention may be used for the research and development of new antiviral drugs, the study of drug resistance to the antiviral drugs, and the study of the pathogenesis of chronic hepatitis B.

[0048] The specific steps for preparing the murine model are as follows:

I. Obtaining Human Stem Cells

[0049] 1. Isolating and culturing human stem cells [0050] 1) Obtaining purified human stem cells. [0051] 2) Culturing and subculturing the stem cells. [0052] 3) Incubating in an incubator at 20 C. to 40 C., 2% to 10% CO.sub.2. [0053] 2. Obtaining commercialized isolated or frozen human stem cells or cell lines.
II. Transplanting stem cells into murines with liver damage [0054] 1. Obtaining different strains of test murine. Examples of test murine include normal mouse, immunodeficient mouse, normal rat, and immunodeficient rat. [0055] 2. Establishing a murine model of liver damage by administering liver-damaging drugs through intraperitoneal injection, intramuscular injection, peripheral intravenous injection, oral administration, or gastric administration, or by partial hepatic resection by surgery. [0056] 3. Transplanting 110.sup.4-8 stem cells by means of peripheral intravenous injection, portal vein injection, spleen injection, or liver injection.
III. HBV infected humanized murines [0057] 1. Injecting each murine with hepatitis B virus through peripheral intravenous injection, subcutaneous injection, intramuscular injection, or intraperitoneal injection. [0058] 2. Detecting the viral load once within 3-30 days after infection of the murines and confirming that the model is established successfully; or detecting the viral load once within 3-30 days after infection of the murines, and testing the viral load in several times to confirm that the model is established successfully.

Definitions

[0059] The pathogenesis of the related hepatitis, liver fibrosis and the like caused by HBV, and the HBV virus clearance are all inflammatory responses mediated by the host immune response. Specific cytotoxic T lymphocytes (CTL), macrophages, dendritic cells and natural killer cells induce liver inflammation and fibrosis. In particular, specific cytotoxic CD8.sup.+ T cells play an important role in the pathogenesis of liver inflammation and viral clearance. Interferon (IFN)- is a product of activated CD8.sup.+ T cells that can induce nitric oxide production to prevent the formation of HBV RNA capsids with replicating ability in hepatocytes in a kinase and proteasome dependent manner This process plays a major role in virus clearance. At the same time, virus-specific CD8.sup.+ T cells migrate to the liver parenchyma and recruit non-antigen-specific polymorphonuclear and mononuclear inflammatory cells, causing hepatocyte apoptosis. A dysfunctional CD8.sup.+ T cell response does not result in IFN- secretion to eliminate HBV and can induce persistently mild damage to hepatocytes and non-parenchymal cell proliferation, leading to chronic HBV infection. Hepatitis B-associated liver fibrosis is a repairing response to inflammatory injury of the liver under HBV stimulation. The activation of liver stellate cells leads to the accumulation of inflammatory cells and the secretion of various cytokines, which eventually leads to excessive deposition of extracellular matrix, and in turn forms liver fibrosis or cirrhosis. However, the specific pathogenesis of chronic hepatitis B, hepatitis B-associated liver fibrosis and liver tumors is still unclear. Accurate research can be performed using the humanized chronic hepatitis B murine model disclosed in the present invention.

[0060] Hepatitis B surface antigen (HBsAg) clearance and hepatitis B secretory antigen (HBeAg) seroconversion are considered to be markers of effective treatment for hepatitis B. The drugs currently approved for the treatment of chronic hepatitis B virus are divided into two categories. The first category is the immunomodulator, including interferon (IFN-) and pegylated interferon . The other category is the nucleoside analog and nucleotide analog prodrug, nucleoside analog including lamivudine, telbivudine and entecavir, nucleotide analog prodrug including adefovir dipivoxil and tenofovir fumarate. However, the ultimate goal in the treatment of hepatitis B is to eliminate nuclear cccDNA or to inhibit the transcriptional activity of cccDNA. The drugs currently used still cannot clear the cccDNA that is latent in the liver. The present invention discloses a humanized chronic hepatitis B murine model constructed based on one type of stem cell technique, which provides a safe and stable preclinical research site for drugs eliminating nuclear cccDNA.

[0061] Currently, the treatment route of liver fibrosis is by inhibiting the inflammatory response of the liver, such as the use of hormones, to block the expression of cytokines; by regulating the TGF- signaling pathway, inhibiting the expression of certain genes, and inhibiting the activation of liver stellate cells, thereby inhibiting the synthesis of the eventual collagen; and by increasing the expression of the metalloproteinase and promoting the degradation of the deposited matrix. However, the commonly used animal models of liver cirrhosis at the present time, such as surgery (such as common bile duct ligation) and chemical drugs (carbon tetrachloride, dimethyl nitrosamine, etc.), are caused by non-hepatitis B virus. Therefore, the mechanism and outcome of the disease are very different from the liver cirrhosis caused by hepatitis B, and they are not suitable for the study of the mechanism and treatment of hepatitis B liver fibrosis and cirrhosis. So far, there are still no drugs with well-defined mechanism for treating this type of disease.

[0062] At present, due to the fact that hepatocellular carcinoma models are mostly caused by chemical drugs or transfection of oncogenes, there are few studies on the mechanism of occurrence, development and treatment of liver tumors caused by hepatitis B. Based on the present model, the occurrence and development of hepatitis B primary liver tumors can be simulated. Through the multi-omics association analysis of proteomics and genomics, the changes in genomics and proteomics in the process of liver tumors metastasis can be found. Identification of genes and proteins with significant differences and analysis of related pathways can reveal the mechanism of liver tumors metastasis. The use of humanized hepatitis B primary liver tumors mouse model to study the mechanism of liver tumors is expected to provide new ideas for the prevention and treatment of liver tumors.

EXAMPLES

[0063] The technical solution of the present invention will be further described below in accordance with the drawings and specific embodiments.

Example 1

[0064] The Research and Development of New Antiviral Drugs Using a Humanized Mouse Model of Chronic Hepatitis B [0065] 1. 150 humanized mice with chronic hepatitis B were randomly divided into three groups with 50 mice in each group. Prior to treatment, the viral load of hepatitis B viruses was about 110.sup.7 and there was no significant difference between the three groups. [0066] 2. Therapeutic dose of a new antiviral drug was administered to the test group. Tenofovir (100 mg/kg), a first-line nucleoside analogue antiviral drug, was orally administered to the control group 1. Antiviral treatment by interferon injection was administered to the control group 2. [0067] 3. At 1 week, 2 weeks, 4 weeks, 8 weeks, 12 weeks and 24 weeks after the treatment, the viral load of HBV in each group of mice was detected, respectively. Adverse effects (such as renal function, myocardial enzymes, etc.) in other major organs were also detected. [0068] 4. The curve of the change in virus load in the test group of mice (i.e., new antiviral drug) was compared with those of the control group 1 (i.e., tenofovir) and the control group 2 (i.e., interferon), thereby obtaining the difference in antiviral effect between the new antiviral drug, the existing first-line antiviral drug tenofovir, and interferon. [0069] 5. The adverse effects of the new antiviral drug were drawn through the observation of side effects, which lays the foundation for conducting a phase 1 clinical trial.

[0070] FIG. 1 is a graph for the development of new antiviral drugs using the humanized mouse model of chronic hepatitis B. It is demonstrated that the humanized mouse model of chronic hepatitis B may be used for the effective screening and testing of new drugs. In the graph, the drop of the viral titer curve of the new drug group is similar to that of the first-line drug tenofovir group, and is superior to the interferon group.

Example 2

[0071] The Study of Efficacy of and Drug Resistance to the Antiviral Drugs Using a Humanized Rat Model of Chronic Hepatitis B [0072] 1. 200 humanized rats with chronic hepatitis B were randomly divided into four groups. There were no significant differences in age, gender, weight and baseline of HBV viral load among the groups. [0073] 2. Therapeutic dose of commonly used antiviral drugs lamivudine, telbivudine, entecavir and tenofovir were administered to each of the four groups, respectively. [0074] 3. At 1 week, 2 weeks, 4 weeks, 8 weeks, 12 weeks and 24 weeks after the treatment, the viral load of HBV in each group of rats was detected, respectively. [0075] 4. Curves were plotted based on the viral load in each group at each time point. The difference between the groups was compared to evaluate the efficacy of and drug resistance to existing commonly used antiviral drugs.

[0076] FIG. 2 is a graph of the viral load in each group for the study of drug resistance to the antiviral drugs using the humanized rat model of chronic hepatitis B. It is demonstrated that the humanized rat model of chronic hepatitis B may be effectively used for the study of drug resistance to the antiviral drugs. In the graph, the drop of the viral titer curves of the tenofovir group and the entecavir group are superior to those of the telbivudine group and the lamivudine group.

Example 3

[0077] The Study of the Pathogenesis of Chronic Hepatitis B Using a Humanized Rat Model of Chronic Hepatitis B [0078] 1. 50 humanized rats with chronic hepatitis B were prepared. The time at which the viruses were injected was defined as the starting point. [0079] 2. On day 1, day 3, day 5, day 7, day 14, day 30, day 60, day 90, day 180 and day 360 from the starting point, 5 rats were sacrificed, respectively. Samples such as serum and liver tissue were collected. [0080] 3. Spectrum changes in the genome and proteome developed due to hepatitis B infection and chronic hepatitis B were identified through multi-omic association analysis such as proteomics and genomics. [0081] 4. The genes and proteins showing significant differences were identified and the relevant pathways were analyzed to reveal the pathogenesis of chronic hepatitis B.

[0082] FIG. 3 is a schematic diagram of the study of the pathogenesis of chronic hepatitis B using the humanized rat model of chronic hepatitis B. From the perspective of proteomics and genomics, such a model may study the mechanism of chronic hepatitis B through analysis of multi-omic association.

Example 4

[0083] The Research and Development of New Anti-Fibrosis Drugs Using a Humanized Rat Model Of Liver Fibrosis and Liver Cirrhosis [0084] 1. When it was clear that inflammation and fibrosis present in the liver of the humanized chronic hepatitis B rat model was liver fibrosis, and when there was characteristic change of pseudo lobule in cirrhosis, this model progressed to the rat model of humanized liver fibrosis and cirrhosis. 75 humanized rats with liver fibrosis and liver cirrhosis were randomly divided into three groups with 25 rats in each group. Prior to treatment, there was no significant difference in the level of liver fibrosis among the three groups. [0085] 2. Therapeutic dose of a new anti-fibrosis drug was administered to the test group. Anluo Huaxian pill and the first-line antiviral drug tenofovir were orally administered to each of the control groups, respectively. [0086] 3. At 1 week, 2 weeks, 4 weeks, 8 weeks, 12 weeks and 24 weeks after the treatment, the level of liver fibrosis in the rats was measured, respectively. Adverse effects (such as renal function, myocardial enzymes, etc.) in other major organs were also detected. [0087] 4. The level of liver fibrosis in the test group of rats (i.e., new antiviral drug) was compared with those in the control groups (i.e., Anluo Huaxian pill and tenofovir), thereby obtaining the difference in anti-fibrosis effect between the new anti-fibrosis drug, Anluo Huaxian pill, and the existing first-line antiviral drug tenofovir. [0088] 5. The adverse effects of the new anti-fibrosis drug were drawn through the observation of side effects, which lays the foundation for conducting a phase 1 clinical trial.

[0089] FIG. 4 demonstrates that the humanized rat model of chronic hepatitis B liver cirrhosis may be used for the effective screening and testing of new drugs. In the graph, the drop of the curve of the level of liver cirrhosis in the new drug group is similar to but slightly better than that in the Anluo Huaxian pill group, and is superior to the first-line drug tenofovir group.

Example 5

[0090] The Study of the Mechanism of Liver Tumors Using a Humanized Mouse Model of Hepatitis B Primary Liver Tumors [0091] 1. When histological and imaging observable primary liver tumors lesions were present in the mouse model of humanized chronic hepatitis B, this model progressed to the mouse model of humanized hepatitis B primary liver tumors. 100 humanized mice with hepatitis B primary liver tumors were fed with conventional feeding with no treatment intervention. [0092] 2. The humanized hepatitis B primary liver tumors model was grouped according to the progress of the tumor metastasis. Mice with single liver tumors lesion only and no metastasis were group into the first group; mice with multiple liver tumors lesions and no metastasis were grouped into the second group; and mice with liver tumors lesions and other organ metastasis were grouped into the third group. There were approximately 25 mice in each group. Samples such as serum, various organ tissues and lymph nodes were collected. [0093] 3. Spectrum changes in the genome and proteome during liver tumors metastasis were identified through multi-omic association analysis such as proteomics and genomics. [0094] 4. The genes and proteins showing significant differences were identified and the relevant pathways were analyzed to reveal the mechanism of liver tumors metastasis.

[0095] As shown in FIG. 5, from the perspective of proteomics and genomics, such a mouse model of liver tumors may be used for studying the mechanism of hepatitis B primary liver tumors through analysis of multi-omic association.

[0096] The above examples are merely the preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Other improvements and changes directly deduced or conceived of by one skilled in the art without departing from the gist and concept of the present invention should also be considered as being included in the scope protected by the present invention.